The long-term goal of the proposed research is to understand the role of cardiolipin in the induction of apoptosis and cardiac dysfunction. Apoptosis has been implicated in the development of cardiac dysfunction associated with aging, ischemia and reperfusion and other pathological conditions. An early event in the development of apoptosis is the loss from the mitochondria of cytochrome c (cyt c), a protein essential to mitochondrial electron transport. Our laboratory has shown that this loss is temporally associated with a decrease in the amount of a mitochondrially located phospholipid, cardiolipin (CL). Using a long chain fatty acid, we induced apoptosis in the cultured neonatal myocyte and demonstrated that the loss of cyt c and CL precedes other measured cellular changes, including the loss of membrane potential. An early event in these sequellae is an inhibition of CL synthesis. Other labs have shown a decrease in CL and loss of cyt c that may be the result of oxidative damage to CL by ROS. These two different systems, one a decrease in cardiolipin synthesis, the other possibly an increase in CL degradation, imply a common role for CL metabolism in the induction of apoptosis. Therefore we hypothesize that a net loss in CL destabilizes membrane association of cyt c, facilitates its release from the mitochondrion and initiates apoptosis. CL levels may be controlled by remodeling of its acyl chain composition, a result of a phospholipase A2 dependent deacylation and acyl chain specific acyltransferase activity. Specific Aim 1 will address the role of CL synthesis and remodeling in at least two models of apoptosis. Since enzymes in the CL biosynthetic and remodeling pathways are potentially regulated by Ca2+, Specific Aim 2 will address the role of Ca2+ CL biosynthetic and remodeling pathways. In Specific Aim 3 we will study the CL/cyt c interaction. CL binds cyt c and may serve to anchor this protein to the outer aspect of the mitochondrial inner membrane.During CL remodeling one or two of the CL acyl chains can be cleaved leaving monolyso-CL which can be reacylated or dilyso-CL which is not a substrate for reacylation. The role of these modified phospholipids in cyt c release will be determined. We will answer the question "is the loss of cyt c preceded by deacylation of CL?" [unreadable] [unreadable]